|BOUTIER, M - University Of Liege
|RONSMANS, M - University Of Liege
|OUYANG, PING - University Of Liege
|FOURNIER, GUILLAUME - University Of Liege
|RESCHNER, ANCA - University Of Liege
|RAKUS, KRZYSZTOF - University Of Liege
|WILKIE, GAVIN - University Of Glasgow
|FARNIR, FRÉDÉRIC - University Of Liege
|BAYROU, CALIXTE - University Of Liege
|LIEFFRIG, FRANÇOIS - University Of Liege
|DESMECHT, D - University Of Liege
|DAVISON, A - Cer Groupe
|VANDERPLASSCHEN, A - University Of Liege
Submitted to: PLoS Pathogens
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/20/2015
Publication Date: 2/20/2015
Citation: Boutier, M., Ronsmans, M., Ouyang, P., Fournier, G., Reschner, A., Rakus, K., Wilkie, G., Farnir, F., Bayrou, C., Lieffrig, F., Li, H., Desmecht, D., Davison, A., Vanderplasschen, A. 2015. Rational development of an attenuated recombinant cyprinid herpesvirus 3 vaccine using prokaryotic mutagenesis and in vivo bioluminescent imaging. PLoS Pathogens. doi: 10.1371/journal.ppat.1004690.
Interpretive Summary: Common carp, and its colorful ornamental variety koi, is one of the most economically valuable species in aquaculture. Since the late 1990s, the common and koi carp culture industries have suffered devastating worldwide losses due to cyprinid herpesvirus 3 (CyHV 3). In the present study, we report the development of an attenuated recombinant vaccine against CyHV-3. Two genes were deleted from the viral genome, leading to a recombinant virus that is no longer capable of causing the disease but can be propagated in cell culture (for vaccine production) and infect fish when added to the water, thereby immunizing the fish. This attenuated recombinant vaccine also had a reduced ability to spread from vaccinated fish to non-vaccinated cohabitant fish. The vaccine induced a protective mucosal immune response capable of preventing the entry of virulent CyHV-3 and is compatible with the simultaneous vaccination of a large number of carp fingerlings by simply immersing the fish in water containing the vaccine. This vaccine represents a promising tool for controlling the most dreadful disease ever encountered by the carp culture industries. In addition, the present study highlights the importance of the CyHV-3 carp model for studying the transmission of Alloherpesviridae and mucosal immunity in teleost skin.
Technical Abstract: Cyprinid herpesvirus 3 (CyHV-3) is causing severe economic losses worldwide in the carp industry, and a safe and efficacious attenuated vaccine compatible with mass vaccination is needed. We produced single deleted recombinants using prokaryotic mutagenesis. When producing a recombinant lacking open reading frame 134 (ORF134), we unexpectedly obtained a clone with additional deletion of ORF56 and ORF57. This triple deleted recombinant replicated efficiently in vitro and expressed an in vivo safety/efficacy profile compatible with use as an attenuated vaccine for mass vaccination of carp fingerlings. To determine the role of each deletion in the phenotype and to further improve the quality of the vaccine candidate, a series of triple, double, and single deleted recombinants were produced and tested in vivo. These experiments identified ORF57 as a major virulence factor and led to the selection of a double deleted recombinant lacking ORF56 and ORF57 as a candidate vaccine. The safety and efficacy of this strain were studied using an in vivo bioluminescent imaging system (IVIS), qPCR, and histopathological examination, which demonstrated that it enters fish via skin infection similar to the wild-type strain. However, compared to the parental wild-type strain, the candidate vaccine replicated at lower levels and spread less efficiently to secondary sites of infection. Transmission experiments allowing water contamination with or without additional physical contact between fish demonstrated that the candidate vaccine has a reduced ability to spread from vaccinated fish to naïve sentinel cohabitants. Finally, IVIS analyses demonstrated that the vaccine candidate induces a protective mucosal immune response at the portal of entry. Thus, the present study is the first to report the rational development of a recombinant attenuated vaccine against CyHV-3 for mass vaccination of carp fingerlings. We also demonstrated the relevance of the CyHV 3 carp model for studying the transmission of Alloherpesviridae and mucosal immunity in teleost skin.